Antisense oligonucleotides targeting Notch2 ameliorate the osteopenic phenotype in a mouse model of Hajdu-Cheney syndrome

Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain–of–function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan. Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix–loop–helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.

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Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels

Molecular and Cellular Biology ◽

10.1128/mcb.01025-08 ◽

2008 ◽

Vol 28 (21) ◽

pp. 6668-6680 ◽

Cited By ~ 61

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Target Genes ◽

Expression Patterns ◽

Activity Levels ◽

Dependent Manner ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions

ABSTRACT The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.

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Regulation of cell survival and proliferation by the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors

Biochemical Society Transactions ◽

10.1042/bst0310292 ◽

2003 ◽

Vol 31 (1) ◽

pp. 292-297 ◽

Cited By ~ 176

Author(s):

K.U. Birkenkamp ◽

P.J. Coffer

Keyword(s):

Transcription Factors ◽

Cell Survival ◽

Cell Fate ◽

Nuclear Export ◽

Molecular Mechanisms ◽

Target Genes ◽

Acute Lymphocytic Leukaemia ◽

Cell Fate Decisions ◽

Forkhead Transcription Factors ◽

Forkhead Box

Recently, the FOXO (Forkhead box, class O) subfamily of Forkhead transcription factors has been identified as direct targets of phosphoinositide 3-kinase-mediated signal transduction. The AFX (acute-lymphocytic-leukaemia-1 fused gene from chromosome X), FKHR (Forkhead in rhabdomyosarcoma) and FKHR-L1 (FKHR-like 1) transcription factors are directly phosphorylated by protein kinase B, resulting in nuclear export and inhibition of transcription. This signalling pathway was first identified in the nematode worm Caenorhabditis elegans, where it has a role in regulation of the life span of the organism. Studies have shown that this evolutionarily conserved signalling module has a role in regulation of both cell-cycle progression and cell survival in higher eukaryotes. These effects are co-ordinated by FOXO-mediated induction of a variety of specific target genes that are only now beginning to be identified. Interestingly, FOXO transcription factors appear to be able to regulate transcription through both DNA-binding-dependent and -independent mechanisms. Our understanding of the regulation of FOXO activity, and defining specific transcriptional targets, may provide clues to the molecular mechanisms controlling cell fate decisions to divide, differentiate or die.

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Tracking the Activation of Stat5 through the Expression of an Inducible Reporter Gene in a Transgenic Mouse Line

Endocrinology ◽

10.1210/en.2011-0053 ◽

2011 ◽

Vol 152 (5) ◽

pp. 1935-1947 ◽

Cited By ~ 6

Author(s):

Nadja Lydia Bednorz ◽

Boris Brill ◽

Andreas Klein ◽

Katrin Gäbel ◽

Bernd Groner

Keyword(s):

Cell Fate ◽

Reporter Gene ◽

Target Genes ◽

Developmental Stages ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Specific Response ◽

Transgenic Mouse Line ◽

Cell Fate Decisions ◽

Apoptosis And Inflammation

Signal transducer and activator of transcription 5 (Stat5), a latent cytoplasmic transcription factor, becomes activated by phosphorylation upon cytokine, hormone, and growth factor interactions with their appropriate receptors and induces the transcription of target genes. It plays crucial roles in principal cell fate decisions and regulates cell differentiation, development, proliferation, apoptosis, and inflammation. It is active in the mammary gland, the liver, hematopoietic cells, and other organs and has pleiotropic functions, depending on its activation pathway and its site of action. We derived transgenic mice in which the expression of a LacZ reporter gene is directed by Stat5-specific response elements and visualized the activation of Stat5 in cells of mouse organs at different developmental stages. The reporter gene activity reflects the timing and the location of Stat5 activation and was documented in mammary epithelial cells during developmental stages of the gland, cells of the liver, kidney, spleen, thymus, and uterus and in granulocytes and macrophages of the transgenic lines.

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MTG16 (CBFA2T3) represses E protein-dependent transcription to regulate colonic secretory cell differentiation, epithelial regeneration, and tumorigenesis

10.1101/2021.11.03.467178 ◽

2021 ◽

Author(s):

Rachel E. Brown ◽

Justin Jacobse ◽

Shruti A. Anant ◽

Koral M. Blunt ◽

Bob Chen ◽

...

Keyword(s):

Cell Differentiation ◽

Mouse Model ◽

Cell Fate ◽

Protein Interactions ◽

Protein Function ◽

Colonic Epithelium ◽

E Proteins ◽

Cell Fate Decisions ◽

Epithelial Regeneration ◽

E Protein

Aberrant epithelial differentiation and regeneration pathways contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 interaction partners include E box-binding basic helix-loop-helix transcription factors (E proteins). MTG16-deficient mice exhibit worse colitis and increased tumor burden in inflammatory carcinogenesis. In this study, we sought to understand the role of MTG16 in colonic epithelial homeostasis and the mechanisms by which MTG16 protects the epithelium in colitis and CAC. We demonstrated that MTG16 deficiency enabled enteroendocrine cell differentiation from secretory precursor cells at the expense of goblet cells. Transcriptomic analysis implicated dysregulated E protein function in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that disrupts MTG16:E protein complex formation (Mtg16P209T), we established that enteroendocrine:goblet cell balance was dependent on MTG16:E protein interactions and that the shift in lineage allocation was associated with enhanced expression of Neurog3, the central driver of enteroendocrine lineage specification. Furthermore, Mtg16 was upregulated in the previously described Ascl2+, de-differentiating cells that replenish the stem cell compartment in response to colon injury. Mtg16 expression was also increased in dextran sulfate sodium (DSS)-treated mouse colon crypts and in IBD patients compared to unaffected controls. We determined that the effects of MTG16 in regeneration are also dependent on its repression of E proteins, as the colonic epithelium failed to regenerate following DSS-induced injury in our novel mutant mouse model. Finally, we revealed that uncoupling MTG16:E protein interactions contributes to the enhanced tumorigenicity in Mtg16-/- colon in the azoxymethane(AOM)/DSS-induced model of CAC. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colonic differentiation and regeneration.

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O-GlcNAc Homeostasis Controls Cell Fate Decisions During Hematopoiesis

10.1101/337881 ◽

2018 ◽

Author(s):

Zhen Zhang ◽

Matt P. Parker ◽

Stefan Graw ◽

Lesya V. Novikova ◽

Halyna Fedosyuk ◽

...

Keyword(s):

Cell Fate ◽

Myeloid Leukemia ◽

Target Genes ◽

Vital Role ◽

Leukemia Cells ◽

Cell Fate Decisions ◽

Expression Of Genes ◽

All Trans Retinoic Acid ◽

Glcnac Transferase

AbstractThe addition of O-GlcNAc (a single β-D-N-acetylglucosamine sugar at serine and threonine residues) by O-GlcNAc transferase (OGT) and removal by O-GlcNAcase (OGA) maintains homeostatic levels of O-GlcNAc. We investigated the role of O-GlcNAc homeostasis in hematopoiesis utilizing G1E-ER4 cells carrying a GATA-1 transcription factor fused to the estrogen receptor (GATA-1ER) that undergo erythropoiesis following the addition of β-estradiol (E2) and myeloid leukemia cells that differentiate into neutrophils in the presence of all-trans retinoic acid. During G1E-ER4 differentiation, a decrease in overall O-GlcNAc levels and an increase in GATA-1 interactions with OGT and OGA were observed. Transcriptome analysis on G1E-ER4 cells differentiated in the presence of Thiamet-G (TMG), an OGA inhibitor, identified expression changes in 433 GATA-1 target genes. Chromatin immunoprecipitation demonstrated that the occupancy of GATA-1, OGT, and OGA atLaptm5gene GATA site was decreased with TMG. Myeloid leukemia cells showed a decline in O-GlcNAc levels during differentiation and TMG reduced the expression of genes involved in differentiation. Sustained treatment with TMG in G1E-ER4 cells prior to differentiation caused a reduction of hemoglobin positive cells during differentiation. Our results show that alterations in O-GlcNAc homeostasis disrupt transcriptional programs causing differentiation errors suggesting a vital role of O-GlcNAcylation in control of cell fate.

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Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

10.1101/2019.12.19.883140 ◽

2019 ◽

Author(s):

Elina Ly ◽

Jennifer F. Kugel ◽

James A. Goodrich

Keyword(s):

Real Time ◽

Cell Fate ◽

Single Molecule ◽

Target Genes ◽

Full Length ◽

Forward Rate ◽

Kinetic Measurements ◽

Response Elements ◽

Cell Fate Decisions ◽

Half Site

AbstractThe tumor suppressor protein p53 is at the nexus of cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds to DNA response elements to regulate transcription of its target genes, a process activated by cellular stress. p53 response elements consist of two decameric half-sites, and most data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding to DNA, unanswered questions remain, due in part to the need for more quantitative and structural studies with the full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding to DNA in real time. The data reveal a dynamic interaction with many p53 binding and dissociation events occurring on single DNA molecules over minutes. We found that p53 tetramers bound to response elements containing only a single half site. The kinetic stability of tetramer/DNA complexes depended on the number of half sites and the helical phasing between them, with the most stable complexes forming on DNA containing two adjacent half sites. The forward rate of binding was not strongly impacted when one half site was mutated. These studies provide real time kinetic measurements of full length p53 tetramers binding to single molecules of DNA, and reveal new insight into the mechanisms by which this nucleoprotein complex forms.

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unc-37/Groucho and lsy-22/AES repress Wnt target genes in C. elegans asymmetric cell divisions

10.1101/2022.01.10.475695 ◽

2022 ◽

Author(s):

Kimberly N. Bekas ◽

Bryan T. Phillips

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Target Gene ◽

Target Genes ◽

Intermediate Cell ◽

Beta Catenin ◽

Cell Fate Decisions ◽

C Elegans ◽

Somatic Gonad ◽

Seam Cell

Asymmetric cell division (ACD) is a fundamental mechanism of developmental cell fate specification and adult tissue homeostasis. In C. elegans, the Wnt/beta-catenin asymmetry (WβA) pathway regulates ACDs throughout embryonic and larval development. Under control of Wnt ligand-induced polarity, the transcription factor TCF/POP-1 functions with the coactivator beta-catenin/SYS-1 to activate gene expression in the signaled cell or, in absence of the coactivator, to repress Wnt target genes in the nascent unsignaled daughter cell. To date, a broad investigation of Groucho function in WβA is lacking and the function of the short Groucho AES homolog, lsy-22 has only been evaluated in C. elegans neuronal cell fate decisions. Further, there is conflicting evidence showing TCF utilizing Groucho-mediated repression may be either aided or repressed by addition of AES subfamily of Groucho proteins. Here we demonstrate a genetic interaction between Groucho repressors and TCF/POP-1 in ACDs in the somatic gonad, the seam hypodermal stem cell lineage and the early embryo. Specifically, in the somatic gonad lineage, the signaled cell fate increases after individual and double Groucho loss of function, representing the first demonstration of Groucho function in wild-type WβA ACD. Further, WβA target gene misexpression occurs at a higher rate than DTC fate changes, suggesting derepression generates an intermediate cell fate. In seam cell ACD, loss of Groucho unc-37 or Groucho-like lsy-22 in a pop-1(RNAi) hypomorphic background enhances a pop-1 seam cell expansion and target gene misregulation. Moreover, while POP-1 depletion in lsy-22 null mutants yielded an expected increase in seam cells we observed a surprising seam cell decrease in the unc-37 null subjected to POP-1 depletion. This phenotype may be due to UNC-37 regulation of pop-1 expression in this tissue since we find misregulation of POP-1 in unc-37 mutants. Lastly, Groucho functions in embryonic endoderm development since we observe ectopic endoderm target gene expression in lsy-22(ot244) heterozygotes and unc-37(tm4649) heterozygotes subjected to intermediate levels of hda-1(RNAi). Together, these data indicate Groucho repressor modulation of cell fate via regulation of POP-1/TCF repression is widespread in asymmetric cell fate decisions and suggests a novel role of LSY-22 as a bona fide TCF repressor. As AES Grouchos are well-conserved, our model of combinatorial TCF repression by both Gro/TLE and AES warrants further investigation.

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Transcription Factor AP4 Mediates Cell Fate Decisions: To Divide, Age, or Die

Cancers ◽

10.3390/cancers13040676 ◽

2021 ◽

Vol 13 (4) ◽

pp. 676

Author(s):

Matthew Man-Kin Wong ◽

Sancy Mary Joyson ◽

Heiko Hermeking ◽

Sung Kay Chiu

Keyword(s):

Transcription Factor ◽

Cell Fate ◽

Cell Cycle Progression ◽

Leucine Zipper ◽

Target Genes ◽

Epithelial Mesenchymal Transition ◽

Ex Vivo ◽

Mesenchymal Transition ◽

Multiple Tumor ◽

Cell Fate Decisions

Activating Enhancer-Binding Protein 4 (AP4)/transcription factor AP4 (TFAP4) is a basic-helix-loop-helix-leucine-zipper transcription factor that was first identified as a protein bound to SV40 promoters more than 30 years ago. Almost 15 years later, AP4 was characterized as a target of the c-Myc transcription factor, which is the product of a prototypic oncogene that is activated in the majority of tumors. Interestingly, AP4 seems to represent a central hub downstream of c-Myc and N-Myc that mediates some of their functions, such as proliferation and epithelial-mesenchymal transition (EMT). Elevated AP4 expression is associated with progression of cancer and poor patient prognosis in multiple tumor types. Deletion of AP4 in mice points to roles of AP4 in the control of stemness, tumor initiation and adaptive immunity. Interestingly, ex vivo AP4 inactivation results in increased DNA damage, senescence, and apoptosis, which may be caused by defective cell cycle progression. Here, we will summarize the roles of AP4 as a transcriptional repressor and activator of target genes and the contribution of protein and non-coding RNAs encoded by these genes, in regulating the above mentioned processes. In addition, proteins interacting with or regulating AP4 and the cellular signaling pathways altered after AP4 dysregulation in tumor cells will be discussed.

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New Insights into the Control of Cell Fate Choices and Differentiation by Retinoic Acid in Cranial, Axial and Caudal Structures

Biomolecules ◽

10.3390/biom9120860 ◽

2019 ◽

Vol 9 (12) ◽

pp. 860 ◽

Cited By ~ 3

Author(s):

Heidrun Draut ◽

Thomas Liebenstein ◽

Gerrit Begemann

Keyword(s):

Retinoic Acid ◽

Gene Family ◽

Cell Fate ◽

Transcriptional Activity ◽

Target Genes ◽

Cell Fate Decisions ◽

Osteogenic Cells ◽

Fin Rays ◽

Important Regulator ◽

Signaling Activity

Retinoic acid (RA) signaling is an important regulator of chordate development. RA binds to nuclear RA receptors that control the transcriptional activity of target genes. Controlled local degradation of RA by enzymes of the Cyp26a gene family contributes to the establishment of transient RA signaling gradients that control patterning, cell fate decisions and differentiation. Several steps in the lineage leading to the induction and differentiation of neuromesodermal progenitors and bone-producing osteogenic cells are controlled by RA. Changes to RA signaling activity have effects on the formation of the bones of the skull, the vertebrae and the development of teeth and regeneration of fin rays in fish. This review focuses on recent advances in these areas, with predominant emphasis on zebrafish, and highlights previously unknown roles for RA signaling in developmental processes.

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Mechanisms in Endocrinology: Notch signaling in skeletal health and disease

Acta Endocrinologica ◽

10.1530/eje-13-0115 ◽

2013 ◽

Vol 168 (6) ◽

pp. R95-R103 ◽

Cited By ~ 42

Author(s):

Stefano Zanotti ◽

Ernesto Canalis

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Target Genes ◽

Skeletal Development ◽

Alagille Syndrome ◽

Notch Signaling Pathway ◽

Loss Of Function ◽

Skeletal Health ◽

Notch Ligand ◽

Ligand Interactions

Notch receptors are single-pass transmembrane proteins that determine cell fate. Upon Notch ligand interactions, proteolytic cleavages release the Notch intracellular domain, which translocates to the nucleus to regulate the transcription of target genes, including Hairy enhancer of split (Hes) and Hes related to YRPW motif (Hey). Notch is critical for skeletal development and activity of skeletal cells, and dysregulation of Notch signaling is associated with human diseases affecting the skeleton. Inherited or sporadic mutations in components of the Notch signaling pathway are associated with spondylocostal dysostosis, spondylothoracic dysostosis and recessive brachydactyly, diseases characterized by skeletal patterning defects. Inactivating mutations of the Notch ligandJAG1or ofNOTCH2are associated with Alagille syndrome, and activating mutations inNOTCH2are associated with Hajdu–Cheney syndrome (HCS). Individuals affected by HCS exhibit osteolysis in distal phalanges and osteoporosis. NOTCH is activated in selected tumors, such as osteosarcoma, and in breast cancer cells that form osteolytic bone metastases. In conclusion, Notch regulates skeletal development and bone remodeling, and gain- or loss-of-function mutations of Notch signaling result in important skeletal diseases.

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